Epigenetic Programming in utero and Later in Life Disease

CNRU Retreat

September 28, 2006

Ken Eilertsen, Ph. D.

Stem Cell Biology Group/Epigenetics and Nuclear Reprogramming

Outline

• Brief overview of where we started and how we got here– How we got here is really a function of the CNRU enabling us

to ask new questions.

• A summary of our first experiment venturing into this area

• Overview of the current P & F award

Past 10 years…

• My lab focused on assisted reproductive technologies research

• Primarily somatic cell nuclear transfer (cloning)– Nuclear reprogramming

• Upon arriving at The Pennington, we wanted to explore new, but very related questions.– Barker Hypothesis– CNRU has been key to meeting this latter objective.

Fetal (Developmental) Origins of Adult Disease Hypothesis:

•Posits that a poor in utero environment elicited by maternal dietaryor placental insufficiency ‘programs’ susceptibility in the fetus to later development of cardiovascular and metabolic disease.

Programming:

-commonly ascribed to any situation where a stimulus or

insult during development establishes a permanent physiological response.

-in the context of a predisposition to a later in life disease,

no mechanism described.

Barker Hypothesis

Some animal models lend support to Developmental Origins of Disease Hypothesis

A.

B.

Maternal low protein diet causes a significant reduction in ICM (embryonic stem cells) and TE (placenta) cell numbers at blastocyststage.

Assisted Reproductive Technologies (ART) in humans have been linked to later in life disease:

• Angelman Syndrome (aberrant DNA methylation)

• Beckwith-Weideman syndrome (aberrant DNA methylation)

Suboptimal culture conditions may be a causative factor forpredisposing offspring to these syndromes

Cause for biallelicexpression was due to aberrant DNA methylation

M Bi

M un-M

Whitte

ns

KSOM

SCNT(cloning)

In vitro fertilization

control

Animals produced by Somatic Cell Nuclear Transfer in particular, can have very profound phenotypes…

Tamashiro et al., Nat Med 8(3), 2002

Cezar et al., Biol Rep 68, 2003

Pace et al., Biol Rep 67, 2002

The general consensus is that the cause of these phenotypes are EPIGENETIC in nature

• Epigenetics is a mechanism that ensures heritable characteristics of cells and functional differences between cell types

• Epigenetic mechanisms alter chromatin (DNA and proteins) in ways that change the availability of genes to transcription factors. Key components include:

– Addition of methyl group to CpG dinculeotides*– Association of Polycomb and other DNA binding proteins that

modify histones.

DNA methylation patterns

• Known to be established during development and subsequently maintained by DNA methyltransferases

• Traditionally thought that once established, the methylation patterns were reliably maintained for the life of the organism and irreversible.

– View is evolving a bit these days.

What is an imprinted gene?• Genes that are mono-allelically expressed

-Either maternal allele OR paternal allele is expressed

• Paternal genes are thought to extract maternal resources for the benefit of the offspring– Growth promoters

• Maternal genes are thought to allocate resources ‘equitably’ between offspring and mother. – Growth suppressors

• Thus, imprinted genes have growth related functions with antagonistic properties.

• Imprinted gene expression is epigenetically regulated.

Are Imprinted Genes Differentially Expressed in Response to Maternal Under-Nutrition?

Facts about Imprinted Genes

• ~70-200 exist in mammals

• Found in clusters (referred to as imprinted domains) at ~ 15 different chromosomal sites

• Imprinted domains are coordinately regulated by epigenetic mechanisms:– DMD, or Differentially Methylated Domain

H19 and Igf2: Examples of imprinted genes and coordinateregulation of expression through a differentially methylated domain.

Experimental design: C57B6 female mice

pcd5 pcd10 pcd19Mating

2 wks priorto mating

C57B6 femalesfed a low protein

Diet.

Pups delivered by C-section;Organs harvested for mRNAisolated to measureexpression levels of imprinted genesin each individual organs (n=10).

Controls fed normal chow diet throughout.

LPD diet

Normal chow diet

Imprinted Genes:

Allele Function (based on observedExpressed phenotypes found in mutations).

Igf2 Paternal Growth Igf2r Maternal Growth retardationH19 Maternal ??Ata3 Paternal Amino acid transport (Placenta)P57kip2 Maternal Reduced survival; growth retardationPeg1/Mest Paternal GrowthPeg3 Paternal Growth

Imprinted Genes:

Liver Brain Heart Kidney

H19

Igf2

Igf2r

Peg3

Ata3

P57kip2

0

0.5

1

1.5

2

2.5

3

3.5

Con Exp

0

0.5

1

1.5

2

2.5

3

Con Exp

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Con Exp

1.4

1.45

1.5

1.55

1.6

1.65

1.7

1.75

1.8

1.85

Con Exp

0

0.5

1

1.5

2

2.5

3

Con Exp

0

0.5

1

1.5

2

2.5

3

Con Exp

0

0.5

1

1.5

2

2.5

3

3.5

Con Exp

0

0.5

1

1.5

2

Con Exp

00.20.40.60.8

11.21.41.61.8

2

Con Exp

ND ND ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

02468

1012141618

Con Exp

Igf2 H19

26 CpG dinucleotides?

Is Altered Expression of H19 Gene Expression Associated with a Change in Methylation of the DMD?

Summary

• Exposure to a maternal LPD during preimplantation period appears to alter imprinted gene expression in organs of near born pups.

• Expression appears to be tissue specific

• No detected differences in methylation patterns of DMD

• Encouraged by the preliminary data indicting imprinted gene expression, known to be regulated epigenetically, could be altered by maternal nutrition.

Submitted a Pilot and Feasibility Grant to the CNRU.

CNRU P & F Proposal

Weaning* 6 wks. 9 wks.* 12 weeks

Normal Chow

Low ProteinPups weaned toHi fat

Low ProteinPups weaned toLow protein

Hi FatPups weaned toHi Fat

Can we link later in life phenotypic characteristics such as weight, BP, glucosetolerance, diabetes,etc. with differentially expressed genes and methylation patterns of associated CpG islands and/or promoters?

Acknowledgements

Ms. Heather KirkDr. Barry Robert (Kidney)Ms. Regina Staten

Stem Cell Biology Lab:Dr. Jeff GimbleDr. Randy MynattDr. Basia KozakDr. Beth Floyd

!Dr. Eric Ravussin!

Dr. Rob Koza:Bisulphite SequencingLow Density Arrays

Microarrays